Compositions and formulations of 9-nitrocamptothecin polymorphs and methods of use therefor

ABSTRACT

A polymorphic form of 9-nitrocamptothecin is provided, the polymorph being characterizable as having, by differential scanning calorimetry, no observable endotherm and an exotherm at between 273.6 and 275.6° C., and a solution NMR spectrum with multiplets at 1.7 and 3.7 ppm shifts.

FIELD OF THE INVENTION

[0001] This invention relates generally to compositions and formulationsof 9-nitro-20-camptothecin polymorphs and methods for their in vivodelivery.

DESCRIPTION OF RELATED ART

[0002] Camptothecin was isolated from the plant, Camptotheca acuminata,in the 1960's (Wall, M. et al. (1966) J. Am. Chem. Soc. 88: 3888-3890).Camptothecin has a pentacyclic ring system with only one asymmetriccenter, in ring E, with a 20(S)-configuration. The pentacyclic ringsystem includes a pyrrole quinoline moiety (rings A, B and C), aconjugated pyridone (ring D), and a six-membered lactone (ring E) withan α-hydoxyl group.

[0003] Camptothecin itself is highly lipophilic and poorly watersoluble. Sodium camptothecin solubilized by sodium hydroxide in waterwas used in clinical trials in the early 1970's and found to haveantineoplastic activity, but as administered intravenously, causedunpredictable side effects such as myelosuppression and hemorrhagiccystitis. Clinical trials with sodium camptothecin were ultimatelydiscontinued because of these toxicities and the lack of consistentantitumor activity.

[0004] Continued evaluation of camptothecin showed that the sodiumcarboxylate salt was only 10% as potent as the native camptothecin withthe closed α-hydroxy lactone ring intact (Wall et al. “InternationalSymposium on Biochemistry and Physiology of the Alkaloids, Mothes et al.eds. (1969) Academic Verlag, Berlin, 77; Giovanella et al. (1991) CancerRes. 51:3052). Studies have demonstrated that camptothecin and itsderivatives undergo an alkaline hydrolysis of the E-ring (α-hydroxylactone, resulting in a carboxylate form of camptothecin, but at pHlevels below 7.0, the α-hydroxy lactone E-ring form of camptothecinpredominates. An intact lactone ring E and α-hydoxyl group have beenshown to be essential for antitumor activity of camptothecin and itsderivatives.

[0005] Camptothecin and its derivatives have been shown to inhibit DNAtopoisomerase I by stabilizing the covalent complex (“cleavablecomplex”) of enzyme and strand-cleaved DNA. Inhibition of topoisomeraseI by camptothecin induces protein-associated DNA single-strand breaks,which occur during the S-phase of the cell cycle. Because the S-phase isshort relative to other cell cycle phases, the toxicity per cell cycleis relatively low, and therefore more acceptable for slowly dividingcells. Exposure to camptothecin for a specified period of time wouldresult in increased cytotoxicity of tumor cells, which divide at a morerapid rate.

[0006] Studies indicate that only the closed α-hydroxy lactone form ofthe drug helps stabilize the cleavable complex, leading to inhibition ofthe cell cycle and apoptosis. To preserve the α-hydroxy lactone form ofcamptothecin, camptothecin and its water insoluble derivatives have beendissolved in N-methyl-2-pyrrolidinone in the presence of an acid (U.S.Pat. No. 5,859,023 to Hausheer et al.). Upon dilution with an acceptableparenteral vehicle, a stable solution of camptothecin was obtained. Theconcentrated solution of camptothecin was also filled in gel capsulesfor oral administration. It is believed that such formulations increasethe amount of lipophilic α-hydroxy lactone form of camptothecin thatdiffuse through the cellular and nuclear membranes in tumor cells.

[0007] Various substituted forms of 20 (S)-camptothecin have beenevaluated for antineoplastic activity. Good activity was found forvarious substitutions to the 20(S)-camptothecin scaffold. For example,9-Amino-20(S)-Camptothecin (“9AC”) and10,11-methylendioxy-20(S)-camptothecin (“10,11, MD”) are capable ofhaving high anticancer activity against human colon cancer xenografts.(Giovanella, B. C., et al., “Highly effective topoisomerase-I-targetedchemotherapy of human colon cancer in xenografts.” (1989) Science246:1046-1048).

[0008] Additionally, 9-nitro-20(S)-camptothecin (referred to herein as“9-nitrocamptothecin” and often abbreviated in the literature as“9-NC”), which has a nine position hydrogen substituted with a nitromoiety, has shown high activity against human tumor xenograft models.9-nitrocamptothecin may be obtained, for example, by extracting thenaturally occurring compound from the plant C. acuminata, according tothe method of Wall et al. (1966), supra, and substituting a nitro moietyfor hydrogen at the nine ring position by known synthetic organicmethods (see for example U.S. Pat. No. 5,922,877 to Cao).9-nitrocamptothecin has inhibited the growth of human tumor xenograftsin immunodeficient nude mice and has also induced regression of humantumors established as xenografts in nude mice with little or noappearance of any measurable toxicity. (D. Chatterjee et al., “Inductionof Apoptosis in Malignant and Camptothecin-resistant Human Cells,”(1996) Annals of the New York Academy of Sciences 803:143). Thus, acontinuing need exists for new and improved ways to exploit the usefultherapeutic activities of 9-nitrocamptothecin and its variousderivatives and analogs.

SUMMARY OF THE INVENTION

[0009] The present invention provides novel polymorphs of9-nitrocamptothecin, including both crystalline and amorphous forms, aswell as pharmaceutical compositions and formulations comprising thesepolymorphs. In one variation, the pharmaceutical compositions andformulations are adapted for administration via oral, injection andinhalation. Various methods are also provided including methods ofmaking the disclosed 9-nitrocamptothecin polymorphs, methods formanufacturing pharmaceutical formulations and compositions comprisingthe polymorphs, as well as methods of using the pharmaceuticalpreparations to treat various diseases.

[0010] In one embodiment, a 9-nitrocamptothecin polymorph is providedthat may be crystallized from acetone or dichloromethane. This polymorphmay be characterized by one or more of the following physicalproperties. The polymorph exhibits an X-ray powder diffraction patternwith salient features being major diffraction lines at °2θ values 8.0and 25.7 for Cu Kα radiation of wavelength 1.5406 Angstrom. Othernotable features of the characterization data of this polymorph include:by differential scanning calorimetry, an endotherm at between about175.5 and 177.5° C., an exotherm at between about 181.7 and 183.7° C.,and an IR spectrum with no absorption centered between about 3625 cm⁻¹and 3675 cm⁻¹.

[0011] In another embodiment, a 9-nitrocamptothecin polymorph isprovided that may be crystallized from tetrahydrofuran. This polymorphmay be characterized by one or more of the following physicalproperties. The polymorph exhibits an X-ray powder diffraction patternwith salient features being major diffraction lines at °2θ values 6.7,12.5, 14.0 and 23.9 for Cu Kα radiation of wavelength 1.5406 Angstrom.Other notable features of the characterization data of this polymorphinclude: by differential scanning calorimetry, no observable endothermand an exotherm at between about 273.6 and 275.6° C., and a solution NMRspectrum with multiplets at about 1.7 and 3.7 ppm shifts.

[0012] In another embodiment, a 9-nitrocamptothecin polymorph isprovided that may be crystallized from acetonitrile. This polymorph maybe characterized by one or more of the following physical properties.The polymorph exhibits, for Cu Kα radiation of wavelength of 1.5406Angstrom, an X-ray powder diffraction pattern with salient featuresbeing major diffraction lines at °2θ values 4.8, 14.2, 19.1 and 26.8.Other notable features of the characterization data of this forminclude: by differential scanning calorimetry, an endotherm at betweenabout 273.9 to 275.9° C., and an exotherm at between about 279.3 and281.3° C.

[0013] In another embodiment, a 9-nitrocamptothecin polymorph isprovided that may be formed by crystallizing 9-nitrocamptothecin fromacetone, dichloromethane, tetrahydrofuran or acetonitrile to form aninitial product, and then recrystallizing the initial product from asolvent comprising a mixture of dimethylformamide and water. Thispolymorph may be characterized by one or more of the following physicalproperties. The polymorph exhibits for Cu Kα radiation of wavelength of1.5406 Angstrom, an X-ray powder diffraction pattern having salientfeatures being major diffraction lines at °2θ values 11.0, 14.0, 16.4and 27.0. Other notable features of the characterization data of thisform include: an IR spectrum with an absorption centered between about3625 cm⁻¹ and 3675 cm⁻¹ and content of more than a trace of water.

[0014] In another embodiment, a 9-nitrocamptothecin polymorph isprovided that may be crystallized from substantially water freedimethyformamide. This polymorph may be characterized by one or more ofthe following physical properties. The polymorph exhibits an X-raypowder diffraction pattern having salient features being diffractionlines at °2θ values 5.4, 10.6 and 26.5 for Cu Kα radiation having awavelength of 1.5406 Angstrom. Other notable features of thecharacterization data of this form include: by differential scanningcalorimetry, an endotherm at between about 149.2 and 151.2° C., anexotherm at between about 162.6 and 164.6° C., and an exotherm atbetween 272 and 274° C.

[0015] Yet other 9-nitrocamptothecin polymorphs are provided that arewholly amorphous or are crystalline with increased amorphous contentcompared to the directly crystallized polymorph as a result of grindingor pulverization. A wholly amorphous polymorph may be obtained by rapidevaporation of solvent from a 9-nitrocamptothecin solution in methanol.This polymorph exhibits no discernable X-ray powder diffraction pattern,comprises a glass. Increased amorphous content polymorphs include thepolymorph obtained by crystallization from ethanol followed by grinding,which exhibits, for Cu Kα radiation of wavelength 1.5406 Angstrom, anX-ray powder diffraction pattern with salient features being majordiffraction lines at °2θ values 10.5 and 12.2, said lines beingbroadened to at least 1.2 times the breadth at half radiation signalamplitude of an X-ray powder diffraction pattern obtained bycrystallizing 9-nitrocamptothecin from ethanol.

BRIEF DESCRIPTION OF THE DRAWINGS

[0016]FIG. 1 illustrates the XRPD pattern of Form A (the “XRPD pattern”being a plot of the intensity of diffracted lines.

[0017]FIG. 2 is a plot of TGA data and the DSC data for Form A.

[0018]FIG. 3 is a plot of the IR absorption spectrum for Form A.

[0019]FIG. 4 is a plot of the Raman absorption spectrum for Form A

[0020]FIG. 5 is a plot of the XRPD pattern of Form B.

[0021]FIG. 6 is a plot of the TGA data and the DSC data for Form B.

[0022]FIG. 7 is a plot of the IR absorption spectrum for Form B.

[0023]FIG. 8 is a plot of the Raman absorption spectrum for Form B.

[0024]FIG. 9 is a plot of the XRPD pattern of Form C.

[0025]FIG. 10 is a plot of the TGA data and the DSC data for Form C.

[0026]FIG. 11 is a plot of the IR absorption spectrum for Form C.

[0027]FIG. 12 is a plot of the Raman absorption spectrum for Form C.

[0028]FIG. 13 is a plot of the XRPD pattern of Form D.

[0029]FIG. 14 is a plot of the TGA data and the DSC data for Form D.

[0030]FIG. 15 is a plot of the IR absorption spectrum for Form D.

[0031]FIG. 16 is a plot of the Raman absorption spectrum for Form D.

[0032]FIG. 17 is a plot of the XRPD pattern of Form E.

[0033]FIG. 18 is a plot of the TGA data and the DSC data for Form E.

[0034]FIG. 19 is a plot of the IR absorption spectrum for Form E.

[0035]FIG. 20 is a plot of the Raman absorption spectrum for Form E.

[0036]FIG. 21 is a plot of the XRPD pattern of Form F.

[0037]FIG. 22 is a plot of the TGA data for Form F.

[0038]FIG. 23 is a plot of the IR absorption spectrum for Form F.

[0039]FIG. 24 is a plot of the Raman absorption spectrum for Form F.

[0040]FIG. 25 is a plot of the XRPD pattern of Form G.

[0041]FIG. 26 is a plot of the TGA data and the DSC data for Form G.

[0042]FIG. 27 is a plot of the IR absorption spectrum for Form G.

[0043]FIG. 28 is a plot of the Raman absorption spectrum for Form G.

[0044]FIG. 29 is a plot of the XRPD pattern of ground9-nitrocamptothecin Form A exhibiting broadened spectral linesindicative of the ground material having increased amorphous contentcompared to unground Form A.

DETAILED DESCRIPTION OF THE INVENTION

[0045] The present invention provides novel polymorphs of9-nitrocamptothecin, including both crystalline and amorphous forms, aswell as pharmaceutical compositions and formulations comprising thesepolymorphs. In one variation, the pharmaceutical compositions andformulations are adapted for administration via oral, injection andinhalation. Various methods are also provided including methods ofmaking the disclosed 9-nitrocamptothecin polymorphs, methods formanufacturing pharmaceutical formulations and compositions comprisingthe polymorphs, as well as methods of using the pharmaceuticalpreparations to treat various diseases.

[0046] Definitions

[0047] As used herein, “amorphous” refers to a material that containstoo little crystal content to yield a discernable pattern by XRPD orother diffraction techniques. Glassy materials are contemplated to beamorphous. Amorphous materials do not have a true crystal lattice, andare consequently glassy rather than true solids, technically resemblingvery viscous non-crystalline liquids. Rather than true solids, glassesmay better be described as quasi-solid amorphous material. Thus anamorphous material refers to a quasi-solid glassy material.Precipitation of a compound from solution, often effected by rapidevaporation of solvent, is known to favor amorphous forms of a compound.

[0048] The term “broad” or “broadened” as used herein to describespectral lines including XRPD, NMR and IR spectroscopy lines is arelative term that relates to the line width of a baseline spectrum. Thebaseline spectrum is often that of an unmanipulated crystalline (definedbelow) form of a specific compound as obtained directly from a given setof physical and chemical conditions, including solvent composition andproperties such as temperature and pressure, for example describing theXRPD spectrum of ground or pulverized crystalline material relative tothe crystalline material prior to grinding. In materials where theconstituent molecules, ions or atoms, as solvated or hydrated, are nottumbling rapidly, line broadening is indicative of increased randomnessin the orientation of the chemical moieties of the compound, thusindicative of an increased amorphous content. When comparisons are madebetween crystalline materials obtained via different crystallizationconditions, broadening indicates either increased amorphous content ofthe sample having the broadened spectral lines, or possibly a mixture ofcrystals that have similar, although not identical spectra.

[0049] As used herein, “crystalline” refers to a material that containsa specific compound, which may be hydrated and/or solvated, and hassufficient crystal content to exhibit a discernable diffraction patternby XRPD or other diffraction techniques. Often, a crystalline materialthat is obtained from a solvent by direct crystallization of a compounddissolved in a solution or interconversion of crystals obtained underdifferent crystallization conditions, will have crystals that containthe solvent, termed a crystalline solvate. Also, the specific solventcomposition and physical properties of crystallization, collectivelytermed crystallization conditions, may result in crystalline materialhaving physical and chemical properties that are unique to thecrystallization conditions. Examples of crystal properties includeorientation of the chemical moieties of the compound with respect toeach other within the crystal and predominance of a specific form of thecompound, for example the lactone form of 9-nitrocamptothecin, which isfavored by the presence of an acid in the solvent composition.

[0050] Depending upon the form of the specific type of crystal present,which dictates the thermodynamic stability of the crystal, variousamounts of amorphous solid material containing the specific compoundwill be present, as a side product of the initial crystallization,and/or a product of degradation of the crystals comprising thecrystalline material. Thus crystalline as used herein contemplatesamorphous content of varying degrees so long as the material has adiscernable diffraction pattern. Often the amorphous content of acrystalline material may be increased by grinding or pulverizing thematerial, which is evidenced by broadening of diffraction and otherspectral lines relative to the unground crystalline material. Sufficientgrinding and/or pulverizing may broaden the lines relative to theunground crystalline material to the extent that the XRPD or othercrystal specific spectrum may become undiscernable, making the materialsubstantially amorphous, or barely discernable, which may be termedquasi-amorphous.

[0051] As contemplated herein the term “trace” refers to an amount thatis detectable by the physical and chemical detection methods employedherein, but comprises less than 0.03 of an equivalent of the specificcompound present in the crystal. For example a crystalline polymorph of9-nitrocamptothecin containing less than 0.04% (w/w) H₂O where a crystalcontaining one H₂O molecule per molecule of 9-nitrocamptothecin, e.g.one equivalent of H₂O, would be approximately 4.4% (w/w) H₂O iscorrectly described as containing a trace of water.

[0052] 1. Polymorphs of 9-Nitrocamptothecin

[0053] Described herein are various polymorphs according to the presentinvention. These polymorphs include crystalline (e.g. true solid) andamorphous (e.g. glassy or quasi-solid) polymorphic forms. For ease,several of the polymorphs described herein are designated A through Gand consistently referenced thereby. In order physically characterizethe polymorphs, various tests were performed including x-ray powderdiffraction (“XRPD”), differential scanning calorimetry (“DSC”),thermogravimetry analysis (“TGA”), hot stage microscopy, infraredspectrometry (“IR”), Raman spectrometry and Karl Fischer analysis. Wherepossible, the results of each test for the different polymorph isprovided.

[0054] A. General Methods of Precipitation, Crystallization,Interconversion Employed in Making 9-Nitrocamptothecin Polymorphs

[0055] Polymorphs according to the present invention may be obtained bydirect crystallization of 9-nitrocamptothecin or by crystallizationfollowed by interconversion. In some instances, the polymorphs arecrystalline and in others amorphous. Amorphous polymorphs may also bederived by rapidly evaporating solvent from solvated9-nitrocamptothecin, or by grinding, pulverizing or otherwise physicallypressurizing or abrading the various crystalline polymorphs describedherein. General organic methods for precipitating and crystallizingorganic compounds may be applied to preparing the various9-nitrocamptothecin polymorphs. These general methods are known to thoseskilled in the art of synthetic organic chemistry and pharmaceuticalformulation, and are described, for example, by J. March, “AdvancedOrganic Chemistry: Reactions, Mechanisms and Structure,” 4th Ed. (NewYork: Wiley-Interscience, 1992).

[0056] B. Form A Polymorph of 9-nitrocamptothecin

[0057] Form A polymorph of 9-nitrocamptothecin may be made bycrystallizing 9-nitrocamptothecin from reagent or HPLC grade ethanol.For example, a saturated or near saturated solution of9-nitrocamptothecin in ethanol may be prepared. The 9-nitrocamptothecinin the saturated or near saturated solution of ethanol may then becrystallized employing conventional methods for crystallizing organiccompounds from organic solvents.

[0058]FIG. 1 illustrates the XRPD pattern of Form A. Major diffractionlines 10 and 12 are observed at approximately 10.5 and 12 °2θrespectively. Sharp, but weaker lines 14, 16 and 18 are observed at13.25, 16, and 24.5 °2θ and several broader and weaker lines areobserved as well.

[0059] Form A exhibits a needle morphology. Consistent with this, mostof the XRPD data on Form A exhibit preferred orientation effects,observed as variations in relative peak intensity, which are oftenobserved in crystalline materials having a needle or plate morphology.

[0060] Thermogravimetric and DSC data on form A is summarized below inTable 1 and plotted in FIG. 2. TABLE 1 Thermal Data on Crystal Form AForm DSC Results* TGA Results** A Endo 278.3, exo 283.9 <0.1

[0061] Making reference now to FIG. 2, no weight loss was observed priorto decomposition above 250° C. A minor exotherm 20 is observed in theDSC curve at a temperature just below the melt endotherm 22 at 278.3° C.and may be due to some decomposition or crystal reordering during orjust prior to the melt. This melt is confirmed by hot stage data,summarized below in Table 2. TABLE 2 Hot Stage Microscopy ObservationsForm Sample # Observations A 1 Needles darken at 210° C., melt onset264° C., melt at 267° C., solid does not recrystallize at RT. A 2Needles, melt onset 262° C., melt with bubble 266° C., solid does notrecrystallize.

[0062] The IR spectrum for Form A is plotted in FIG. 3. The spectrumshows a relatively sharp OH stretch 30 around 3430 cm⁻¹, aromatic andaliphatic CH stretches 32 between 3100 and 2800 cm⁻¹, and a very complexfingerprint region 34 from 1700-400 cm⁻¹.

[0063] The Raman spectrum for Form A is provided in FIG. 4. The Ramanspectrum shows relatively weak aromatic and aliphatic CH stretches 40between 3100 and 2800 cm⁻¹, and stronger bands 42 in the region from1700-1300 cm⁻¹.

[0064] Data of moisture sorption/desorption for Form A are summarizedbelow in Table 3. TABLE 3 Moisture Sorption/Desorption Data for Form A.Samp Elap Time Weight Weight % Temp Samp Min Mg chg Deg C. RH % 0.012.3086 0.0000 24.99 14.41 11.1 12.3102 0.0131 25.02 5.16 19.6 12.31040.0149 25.02 15.27 27.2 12.3107 0.0169 25.03 24.98 34.7 12.3112 0.021025.03 35.07 42.2 12.3116 0.0242 25.03 45.07 50.2 12.3121 0.0283 25.0355.00 59.2 12.3125 0.0315 25.03 64.96 68.3 12.3129 0.0347 25.03 74.7577.3 12.3134 0.0387 25.03 84.64 86.3 12.3138 0.0421 25.03 94.90 94.812.3139 0.0429 25.03 84.95 103.8 12.3139 0.0429 25.03 75.18 111.812.3136 0.0405 25.02 65.02 119.8 12.3133 0.0381 25.02 54.99 127.812.3132 0.0372 25.02 45.05 135.8 12.3129 0.0348 25.02 34.97 143.312.3126 0.0324 25.02 24.95 150.3 12.3124 0.0307 25.03 15.00 157.312.3120 0.0275 25.03 4.83

[0065] Form A gains a minimal amount of water (0.01%) upon equilibrationto 5% RH. The material gained a total of only about 0.03% in the regionfrom 5 to 95% RH, indicating that the material is not hygroscopic. Asmall amount of hysteresis exists between the hydration and dehydrationcurves. The characterization data obtained for Form A show that it is acrystalline, unsolvated material, which is not hygroscopic and melts atabout 278° C.

[0066] C. Form B Polymorph of 9-nitrocamptothecin

[0067] Form B polymorph of 9-nitrocamptothecin may be made bycrystallizing 9-nitrocamptothecin from reagent or HPLC grade acetone ordichloromethane. For example, a saturated or near saturated solution of9-nitrocamptothecin in acetone or dichloromethane is prepared. Thesaturated or near saturated solution of 9-nitrocamptothecin in acetoneor dichloromethane is then crystallized from solution employingconventional methods for crystallizing organic compounds from organicsolvents.

[0068] The XRPD pattern of Form B is provided in FIG. 5. The XRPDpattern of Form B has two major diffraction lines 50 and 52 at about 8.0and 25.7 °2θ respectively. There are also several additional broad, weaklines. The XRPD pattern tends to indicate that Form B is not highlycrystalline.

[0069] An XRPD pattern was also obtained from a sample of Form B thatwas kept in a closed vial for 31 days at ambient conditions, whichpattern did not differ from FIG. 5, indicating the Form B crystallinepolymorph is relatively stable. The remaining characterization data,discussed below, suggest that this Form B is a hemihydrate of9-nitrocamptothecin.

[0070] Thermogravimetric and DSC data on Form B are provided below inTable 4 and plotted in FIG. 6. TABLE 4 Thermal Data on Crystal Form BForm DSC Results* TGA Results** B endo 68.7, 176.5 1.3 exo 182.7, 265.4

[0071] The data show a gradual weight loss of 1.3% out to 200° C. withdecomposition occurring above 225° C. The result of a Karl Fischer wateranalysis for Form B was 2.66%. This tends to demonstrate that Form Bcontains approximately 2.7% water by weight, and is thus hydrated. Anydifference between the TGA and the Karl Fischer analysis may be due towater loss while the sample was equilibrating at 35° C. at the start ofthe TGA experiment. The theoretical amount for a monohydrate would beapproximately 4.4%. Based on the Karl Fischer data, it appears that FormB is a hemihydrate.

[0072] The DSC curve for Form B is provided in FIG. 6. It shows a broadendotherm 60 at 68.7° C., assigned to loss of water. This is followed bya sharp endotherm 62 at 176.5° C. and an exotherm 64 at 182.7° C.,assigned to a possible melt/recrystallization. The hot stage microscopydata are summarized in Table 5. TABLE 5 Hot Stage MicroscopyObservations Form Sample # Observations B 1 Needles, melt onset 262° C.,melt with bubbles at 266° C., solid does not recrystallize.

[0073] The hot stage microscopy data indicates a change in birefringenceat 159° C., but a clear melt/recrystallization was not observed underthese conditions. The hot stage also showed a melt onset at 200° C. Thiswas not clearly observed in the DSC, which shows a broad exotherm 66with a maximum 68 at 265.4° C., and is probably due to energeticdecomposition observed in this temperature range.

[0074] The IR spectrum for Form B is provided in FIG. 7. The IR spectrumdemonstrates a relatively broad OH stretch 70 around 3400 cm⁻¹. Thearomatic and aliphatic CH stretches 72 between 3100 and 2800 cm⁻¹ arebroadened compared to the stretches for Form A, as well as the complexfingerprint region 74 from 1700-400 cm⁻¹, which includes at least onenew absorbance at 1710 cm⁻¹.

[0075] The Raman spectrum for Form B is provided in FIG. 8. The Ramanspectrum shows relatively weak aromatic and aliphatic CH stretches 80between 3100 and 2900 cm⁻¹, and stronger bands 82 in the region from1700-1200 cm⁻¹.

[0076] Moisture sorption/desorption data for Form B is provided below inTable 6. TABLE 6 Moisture Sorption/Desorption Data for Form B. Samp ElapTime Weight Weight % Temp Samp Min Mg chg deg C. RH % 0.0 1.3612 0.000025.03 9.87 27.0 1.3478 −0.9820 25.04 5.27 48.0 1.3545 −0.4898 25.0414.99 66.0 1.3596 −0.1151 25.04 24.85 79.3 1.3636 0.1788 25.04 35.1091.3 1.3670 0.4286 25.04 45.00 102.9 1.3699 0.6416 25.04 54.97 115.81.3725 0.8314 25.04 64.84 129.0 1.3746 0.9869 25.04 74.71 142.1 1.37631.1118 25.04 84.56 158.8 1.3779 1.2293 25.04 94.72 167.2 1.3771 1.170625.04 85.04 182.0 1.3752 1.0310 25.03 75.38 195.4 1.3727 0.8473 25.0364.98 209.1 1.3702 0.6637 25.04 55.12 222.0 1.3671 0.4359 25.04 45.03235.1 1.3637 0.1861 25.04 34.99 269.9 1.3596 −0.1151 25.04 24.89 295.81.3542 −0.5118 25.04 14.94 321.7 1.3470 −1.0408 25.04 5.00

[0077] This data indicates that Form B lost a significant amount ofwater (0.98%) upon equilibration at 5% RH. The material then gained atotal of 2.21% in the region from 5 to 95% RH, indicating that thematerial is relatively hygroscopic. There is essentially no hysterisisbetween the hydration and dehydration curves. Based on thecharacterization data, Form B appears to be a hemihydrate of9-nitrocamptothecin, which can gain or lose water with minimal change inthe crystal form.

[0078] D. Form C Polymorph of 9-nitrocamptothecin

[0079] Form C polymorph of 9-nitrocamptothecin may be made bycrystallizing 9-nitrocamptothecin from reagent or HPLC gradetetrahydrofuran. For example, a saturated or near saturated solution of9-nitrocamptothecin in tetrahydrofuran is prepared. The saturated ornear saturated solution of 9-nitrocamptothecin in tetrahydrofuran isthen crystallized from solution employing conventional methods forcrystallizing organic compounds from organic solvents.

[0080] The XRPD pattern of Form C is provided in FIG. 9. The Form Cpattern has major diffraction lines 90, 92, 94, 96 at about 6.7, 12.5,14.0 and 23.9 °2θ respectively. The XRPD pattern indicates that thematerial is not highly crystalline and may contain some amorphousmaterial. This pattern was only obtained on samples generated fromtetrahydrofuran.

[0081] TGA data on Form C are provided below in Table 7. TABLE 7 ThermalData on Crystal Form C Form DSC Results* TGA Results** C exo 134.1,156.5, 274.6 15.7

[0082] The data show a weight loss of 15.7% below 200° C. withdecomposition occurring above 250° C. This is comparable to thetheoretical amount (15.5%) for a mono THF solvate. The characterizationof this weight loss as THF solvent is supported by the ¹H NMR data. Anattempt to desolvate this form under vacuum, indicated this form becomesamorphous under these conditions.

[0083] Thermogravimetric and DSC data for Form C is plotted in FIG. 10.No clear endothermic transitions corresponding to loss of solvent orwater are observed. However, a broad exotherm 100 at 156.5° C. isobserved with a shoulder 102 at 134.1° C. Based on hot stage microscopy,this may correspond to a recrystallization which occurs as the solventleaves. Again, a melt (endotherm) is not observed prior to thedecomposition exotherm at 274.6° C., however, a melt is evident by hotstage microscopy. This indicates that the melt endotherm is not evidentby DSC due to it's being marked by the highly energetic decomposition.Hot stage microscopy results are summarized below in Table 8. TABLE 8Hot Stage Microscopy Observations for Form C Form Sample # ObservationsC 1 Opaque solids, small solids melt at 126° C., melt onset 246° C.,melt at 263° C.

[0084] The IR spectrum for Form C is provided in FIG. 11. The IR datacollected on this form show an extremely broad OH stretch 110 around3400 cm⁻¹. No clear absorption bands due to THF can be assigned.Although the peak positions generally correspond with those of Form A,all the peaks in the entire spectrum have broadened significantly. TheRaman spectrum for Form C is provided in FIG. 12. The Raman spectrumshows relatively weak aromatic and aliphatic CH stretches 120 between3100 and 2800 cm⁻¹, and stronger bands 122 in the region from 1700-1300cm⁻¹.

[0085] The result of a Karl Fischer water analysis was 1.06%. Theseresults indicate that Form C contains approximately 1.1% water byweight, which may be associated with the amorphous portion of thesample. Based on the characterization data, Form C appears to be apoorly crystalline THF solvate, which desolvates to a mainly amorphousmaterial.

[0086] E. Form D Polymorph of 9-nitrocamptothecin

[0087] Form D polymorph of 9-nitrocamptothecin may be made bycrystallizing 9-nitrocamptothecin from reagent or HPLC gradeacetonitrile. For example, a saturated or near saturated solution of9-nitrocamptothecin in acetonitrile is prepared. The saturated or nearsaturated solution of 9-nitrocamptothecin in acetonitrile is thencrystallized from solution employing conventional methods forcrystallizing organic compounds from organic solvents.

[0088] The XRPD pattern of Form D is provided in FIG. 13. The Form Dpattern has major diffraction lines 130, 132, 134, 136 at about 4.8,14.2, 19.1 and 26.8 °2θ respectively. Several additional broad, weaklines are observed as well. This pattern was only obtained from materialin contact with acetonitrile.

[0089] TGA data on Form D are summarized below in Table 9. TABLE 9Thermal Data on Crystal Form D Form DSC Results* TGA Results** D Endo177.6, 274.9 8.7 exo 201.2, 280.3

[0090] This data shows a weight loss of 8.7% below 200° C. withdecomposition occurring above 250° C. This is comparable to thetheoretical amount (9.4%) for a mono acetonitrile solvate. A KarlFischer water analysis indicates form D contains less than 0.9% water byweight. These data confirm that the TGA observed weight loss is due toacetonitrile.

[0091] The DSC curve for Form D is provided in FIG. 14. It shows a broadendothermic transition 140 corresponding to loss of solvent at 177.6° C.This is consistent with the hot stage data, summarized in Table 10below, which illustrated solids darkening at 183° C. TABLE 10 Hot StageMicroscopy Observations for Form D Form Sample # Observations D 1Plates, lose birefringence at 180° C., some recrystallization at 241°C., melt onset 262° C.

[0092] Making reference now to FIG. 14, the broad endothermic transition140 is followed by a broad exotherm 142 at 201.2° C. This likelycorresponds to a recrystallization, which occurs as the solvent leavesand would be consistent with the hot stage observation at 244° C. ofsmall needles growing. A small melt endotherm 144 is observed at 274.9°C., just prior to the decomposition exotherm 146 at 280.3° C., whereasthe melt onset is observed at 262° C. by hot stage microscopy.

[0093] The IR spectrum is plotted in FIG. 15. The IR data on Form D showan extremely broad OH stretch 150 shifted to around 3265 cm⁻¹ and asmaller sharp resonance 152 around 3470 cm⁻¹. Two sharp resonances 154and 156 are observed for the acetonitrile CN stretch at 2253 and 2290cm⁻¹. All the peaks in the entire spectrum have broadened significantlyfrom that of Form A.

[0094] The Raman spectrum is plotted in FIG. 16. The Raman spectrumshows relatively weak aromatic and aliphatic CH stretches 160 between3100 and 2800 cm⁻¹, and stronger bands 162 in the region from 1700-1300cm⁻¹. There is also a CN resonance 164 at 2250 cm⁻¹. Based on thecharacterization data, Form D is a crystalline acetonitrile solvate,which may change forms with heating and/or solvent loss.

[0095] F. Form E Polymorph of 9-nitrocamptothecin

[0096] Form E polymorph of 9-nitrocamptothecin may be made bycrystallizing 9-nitrocamptothecin from reagent or HPLC grade chloroform.For example, a saturated or near saturated solution of9-nitrocamptothecin in chloroform is prepared. The saturated or nearsaturated solution of 9-nitrocamptothecin in chloroform is thencrystallized from solution employing conventional methods forcrystallizing organic compounds from organic solvents.

[0097] The XRPD pattern of Form E is provided in FIG. 17. It has majordiffraction lines 170 and 172 at about 7.4 and 22.5 °2θ respectively.Some of the XRPD patterns obtained on Form E show additional broad linesat 8.1 and 20.9 °2θ. These additional lines may be due to preferredorientation effects. The XRPD pattern was only obtained from chloroformand indicates that the material is not highly crystalline.

[0098] TGA data on Form E is summarized below in Table 11. It shows aweight loss of 8.5% below 200° C. with decomposition occurring above250° C. This corresponds to approximately 0.3 mole of chloroform permole of 9-nitrocamptothecin whereas the theoretical amount for a singleequivalent of chloroform solvent is 23.3%. TABLE 11 Thermal Data onCrystal Form E Form DSC Results* TGA Results** E Endo 276.2 8.5 exo181.6, 200.9, 281.9

[0099] Results of a Karl Fischer water analysis were less than 0.67%.These results indicate that Form E contains less than approximately 0.7%water by weight. Thus, Form E appears to contain approximately onequarter of a mole, e.g. between one fifth and one third of anequivalent, of chloroform solvent.

[0100] The DSC curve is provided in FIG. 18. There are two exothermictransitions, which occur at slightly higher temperatures than that forForm C. No clear endothermic transitions corresponding to loss ofsolvent or water are observed. However, two broad exotherms 180 and 182are observed at 181.6° C. and 200.9° C. These transitions may correspondto recrystallizations occuring as the solvent is depleted.

[0101] This is consistent with the hot stage microscopy data summarizedbelow in Table 12, which indicate small needles melting at 197° C., thenneedles growing again at 239° C. In contrast to Form C, a small meltendotherm 184 is observed at 276.2° C., just prior to the sharpdecomposition exotherm 186 at 281.9° C. This is supported by the hotstage data, which shows needles finally melting at 265° C. TABLE 12 HotStage Microscopy Observations for Form E Form Sample # Observations E 1Small opaque needles, melt at 194° C., recrystallization at 236° C.,melt onset 262° C.

[0102] The IR spectrum for Form E is plotted in FIG. 19. The carbonylstretch 190 at 1710 cm⁻¹ is shifted from that for Form B. No additionalpeaks due to the chloroform solvent were observed. Although the peakpositions generally correspond with those of form A, all the peaks inthe entire spectrum have broadened significantly.

[0103] The Raman spectrum for Form E is provided in FIG. 20. The Ramanspectrum shows relatively weak aromatic and aliphatic CH stretches 200between 3100 and 2800 cm⁻¹, and stronger bands 202 in the region from1700-1300 cm⁻¹. Based on the characterization data, Form E is a poorlycrystalline material that may be a one-quarter chloroform solvate.

[0104] G. Form F Polymorph of 9-nitrocamptothecin

[0105] Form F polymorph of 9-nitrocamptothecin may be made bycrystallizing 9-nitrocamptothecin from reagent or HPLC grade ethanol,acetone, dichloromethane, tetrahydrofuran or acetonitrile to form aninitial crystalline product as described for Forms B, C and D, andrecrystallizing the initial crystalline product from a saturatedsolution of dimethylformamide (DMF) and water (DMF/H2O :: 75%/25%, v/v)that contains excess undissolved material comprising the initialcrystalline material. The recrystallization from a saturated solutioncontaining an initital crystalline material is known as aninterconversion. The solubility of 9-nitrocamptothecin in DMF/H2O ::75%/25%, v/v is then determined. A saturated solution of9-nitrocamptothecin in DMF/H2O :: 75%/25%, v/v having a visible excessof the initial crystalline material, e.g. a slurry, is then prepared.The saturated solution of 9-nitrocamptothecin in DMF/H2O :: 75%/25%, v/vslurry of the initial crystalline material is then recrystallized(interconverted) from solution. This recrystallization from the slurryis carried out by agitating the slurry for 7 days at ambient temperatureusing a shaker block or rotating wheel. After the 7-day agitation,insoluble solids are recovered from the slurry by filtration. Therecrystallization or interconversion is obtained over a relatively longperiod of time relative to crystallization of the initial crystallineproduct. Again, as it is known in the art of growing crystals in generalthat higher quality crystals are generally favored by conditions thatfavor slower crystal formation, including those affecting kineticsdirectly, such as temperature, the recrystallization conditions may beappropriately be adjusted to obtain higher quality crystalline materialas necessary. Thus, for example, if poor crystals are formed under aninitial set of interconversion conditions, the solvent temperature maybe reduced and the agitation period can be decreased relative to theinitial set of interconversion conditions.

[0106] The XRPD pattern of Form F is provided in FIG. 21. It has majordiffraction lines 210, 212, 214, and 216 at about 11.0, 14.0, 16.4 and27.0 °2θ respectively. Several additional sharp, but weaker, lines areobserved as well. The pattern indicates that the material is relativelyhighly crystalline. This pattern was obtained from material from aninterconversion study using DMF/H₂O (75%/25%, v/v) as the solvent.

[0107] TGA data on Form F are summarized below in Table 13 and providedin FIG. 22. The data show a weight loss of only 1.2% between 100 and230° C., with decomposition occurring above 250° C. Referring to FIG.22, the TGA curve for Form F is similar to that observed for thehydrated form B. The theoretical amount for a single equivalent of wateris approximately 4.4%. TABLE 13 Thermal Data on Crystal Form F Form TGAResults* F 1.2

[0108] The IR spectrum for Form F is plotted in FIG. 23. The IR datashow an extremely broad OH stretch 230 around 3400 cm⁻¹ and a sharp,crystalline OH stretch 232 at 3650 cm⁻¹.

[0109] The Raman spectrum for Form F is provided in FIG. 24. The Ramanspectrum shows relatively weak aromatic and aliphatic CH stretches 240between 3100 and 2800 cm⁻¹, and stronger bands 242 in the region from1700-1300 cm⁻¹. The characterization data are consistent with thedescription of this form as a hydrate containing between one fourth andone third of an equivalent of water.

[0110] H. Form G Polymorph of 9-nitrocamptothecin

[0111] Form B polymorph of 9-nitrocamptothecin may be made bycrystallizing 9-nitrocamptothecin from reagent or HPLC gradedimethylformamide (reagent or HPLC grade DMF being substantially waterfree, defined as containing no more than a trace of water). For example,a saturated or near saturated solution of 9-nitrocamptothecin in DMF isprepared. The saturated or near saturated solution of9-nitrocamptothecin in DMF is then crystallized from solution employingconventional methods for crystallizing organic compounds from organicsolvents.

[0112] As it is well appreciated in the art of growing crystals ingeneral that higher quality crystals, e.g. forming crystals having fewerlattice defects and proportionately less glassy material, are generallyfavored by conditions that favor slower crystal formation, includingthose slowing solvent evaporation and those affecting kineticsgenerally, the crystallization conditions may be appropriately beadjusted to obtain higher quality crystalline material as necessary.Thus, for example, if poor crystals are formed under an initial set ofcrystallization conditions, the solvent temperature may be reduced andambient pressure above the solution may be increased relative to theinitial set of crystallization conditions.

[0113] The XRPD pattern of Form G is provided in FIG. 25. It has majordiffraction lines 250, 252 and 254 at about 5.4, 10.6 and 26.5 °2θrespectively. A few additional broad lines are observed as well. Thispattern was obtained from DMF.

[0114] TGA data on Form G is summarized below in Table 14. It shows arather gradual weight loss of 0.9% out to approximately 135° C.,followed by a significant weight loss of 5.5% out to 235° C., withdecomposition occurring above this temperature. The theoretical amountfor a single equivalent of DMF solvent is approximately 15.7%. TABLE 14Thermal Data on Crystal Form G Form DSC Results* TGA Results** G endo82.2, 150.2 6.4 exo 163.6, 273.0

[0115] The DSC curve for Form G is provided in FIG. 26. It is similar tothe DSC curve for Form B and shows a broad endotherm 260 at 82.2° C.This broad endotherm may be assigned to loss of volatiles and isfollowed by a sharp endotherm 262 at about 150.2° C. and an exotherm 264at 163.6° C., assigned to either a solvent loss or a melt followed byrecrystallization. A melt endotherm is not observed prior to thedecomposition exotherm 266 at about 273° C. This data is consistent witha mixed hydrate/solvate form.

[0116] The IR spectrum for Form G is plotted in FIG. 27 and the Ramanspectrum is plotted in FIG. 28. The Raman spectrum shows relatively weakaromatic and aliphatic CH stretches 280 between 3100 and 2800 cm⁻¹, andstronger bands 282 in the region from 1700-1300 cm⁻¹. As characterizedby the preceding data, Form G appears to be a relatively poorlycrystalline mixed hydrate/solvate polymorph of 9-nitrocamptothecin.

[0117] I. Amorphous or Increased Amorphous Content Material

[0118] The XRPD data for the increased amorphous content materialobtained by grinding a crystalline polymorph are plotted in FIG. 29.This material is obtained from grinding Form A. Ground Form A has adiscernable XRPD pattern, and is therefore crystalline as definedherein. Ground Form A exhibits a discernable broadening of the Form AXRPD pattern, indicative of increased amorphous content compared to FormA as crystallized from ethanol. Further grinding is expected to increasethe amorphous content and further broaden the XRPD pattern with thelimit of the XRPD pattern being so broadened that it cannot be discernedabove noise. When the XRPD pattern is broadened to the limit of beingindiscernable, the material is no longer a crystalline material havingincreased amorphous content relative to unground crystallized material,but is a material that is wholly amorphous as defined herein. Althoughthe peaks that are present are consistent with form A material they arebroadened considerably along with the appearance of an amorphous haloindicating that the material is losing crystallinity after grinding Nopeaks were observed that would indicate grinding produces another form.

[0119] Amorphous material was also obtained after rapidly removingmethanol by evaporation from a 9-nitrocamptothecin in methanol solutionon a rotovap. The XRPD data for the amorphous material (wholly amorphousas defined herein, and denoted Form X) derived from rapid evaporation of9-nitrocamptothecin dissolved in methanol is provided in FIG. 29.

[0120] 2. Formulations and Administration Modalities

[0121] The present invention also encompasses pharmaceuticalformulations comprising one or more 9-nitrocamptothecin polymorphs ofthe present invention and a pharmaceutical carrier or diluent, whereinthe 9-nitrocamptothecin remains in its polymorphic form.

[0122] Formulations according to the present invention may be adaptedfor any type of administration where it is feasible to administer solidparticles of a drug. For example, the formulatins can be administeredorally, parenterally, intraperitoneally, intravenously, intraarterially,transdermally, sublingually, intramuscularly, rectally, transbuccally,intranasally, liposomally, via inhalation, vaginally, intraoccularly,via local delivery (for example by catheter or stent), subcutaneously,intraadiposally, intraarticularly, or intrathecally, optionally in aslow release dosage form. In particular embodiments, the9-nitrocamptothecin polymorphs are administered orally, by inhalation orby injection subcutaneously, intramuscularly intravenously or directlyinto the cerebrospinal fluid.

[0123] A. Oral and Parenteral Formulations

[0124] According to one embodiment, one or more polymorphic forms may beformulated for oral administration. The concentration of the polymorphsgiven in any oral formulation is determined by the final desiredformulation. The total amount of all polymorphs present in theformulation is preferably an amount that will allow a recommended doseto be conveniently administered. One factor in determining the amount ofthe polymorph or polymorphs contained in an oral dose is the requiredsize of the delivery vehicle.

[0125] Solid dosage forms for oral administration include capsules,tablets, pills, powders, and granules. In solid dosage forms, the activeagent is admixed with at least one inert pharmaceutically acceptablecarrier such as sucrose, lactose, or starch. Such dosage forms can alsocomprise, as is normal practice, an additional substance other than aninert diluent, e.g., a lubricating agent such as magnesium stearate.With capsules, tablets, and pills, the dosage forms may also comprise abuffering agent. Tablets and pills can additionally be prepared withenteric coatings.

[0126] Liquid dosage forms for oral administration includepharmaceutically acceptable, suspensions and syrups, with the elixirscontaining an inert diluent commonly used in the art, such as water.These compositions can also include one or more adjuvants, such as asurface stabilizing agent, a suspending agent, a sweetening agent, aflavoring agent or a perfuming agent. 9-nitrocamptothecin is maintainedin any disclosed polymorph form when the invention is embodied as aliquid dosage form.

[0127] According to this aspect, the 9-nitrocamptothecin polymorph ismixed with other compounds or delivery devices to form stablecompositions with enhanced therapeutic activity. These formulationspermit oral administration to tumor-bearing subjects, such as humanpatients with cancer. For example, in one embodiment, the9-ntirocamptothecin polymorph forms may be mixed with pharmaceuticallyacceptable powdered excipients, carriers and/or diluents. Thecompositions and amount of each additional material in the formulationwill depend upon various factors, including, the speed ofadministration, the timing of drug delivery after administration of theformulation and final desired concentration. Examples of exicipents thatmay be included in such formulations include a pH adjustment compound,typically either a pharmaceutically acceptable acid or base, and/or abuffering agent, comprising approximately equimolar ratio of a weak acidor base and the conjugate salt thereof.

[0128] In one embodiment, the formulation may comprise a polymorphcombined with a surface interaction inhibitor, which creates a physicalbarrier between adjacent particles. In this formulation, the9-nitrocamptothecin is preferably a crystalline polymorph (e.g. a truesolid) having a relatively small particle size, which is expected tostabilize the 9-nitrocamptothecin better than a glassy or amorphous,quasi-solid material having the same particle size. The small yet stableparticles 9-nitrocamptothecin delivered in this composition are expectedto have better bioavailability and higher therapeutic activity whenadministered orally compared to dosage forms having larger particlesize, while having a longer shelf life than preparations comprisingsmall glassy particles.

[0129] Preparations for parenteral administration include sterileaqueous or non-aqueous suspensions, and microsuspensions. Examples ofnon-aqueous vehicles are propylene glycol, polyethylene glycol,vegetable oils, such as olive oil and corn oil, gelatin, and injectableorganic esters such as ethyl oleate. Those of skill in the art offormulating pharmaceutical preparations will appreciate that completesolvation of crystalline or amorphous solids is not encompassed by theinstant invention and the polymorph should be insoluble in the carrierto preserve the polymorph that is to be employed in the specificformulation. Such dosage forms may also contain one or more adjuvantssuch as a preserving agent, for example a surface interaction inhibitor,a wetting agent and a dispersing agent. The dosage forms may besterilized by, for example, filtration through a bacteria-retainingfilter, by incorporating sterilizing agents into the compositions, byirradiating the compositions, or by heating the compositions. They canalso be manufactured using sterile water, or some other sterileinjectable medium, prior to use.

[0130] Pharmaceutical formulations for oral or parenteral administrationmay also comprise a 9-nitrocamptothecin polymorph-containingmicrosuspension, and may contain alternative pharmaceutically acceptablecarriers, vehicles, additives, etc. particularly suited to oral orparenteral drug administration. Alternatively, a 9-nitrocamptothecinpolymorph-containing microsuspension may be administered orally orparenterally without modification. Microsuspensions arethermodynamically stable dispersions of microcrystals, which may bestabilized by an interfacial film of surfactant molecules functioning asa dispersing agent (Encyclopedia of Pharmaceutical Technology (New York:Marcel Dekker, 1992), volume 9).

[0131] B. Pulmonary Administration

[0132] Any of the 9-nitrocamptothecin polymorphs may be employed forpulmonary administration. Both crystalline polymorphs, wherein thecrystals are true solid materials, and wholly amorphous, glassy,quasi-solid polymorphs lend themselves to being rendered to anappropriate particle size for both dry and aerosolized liquid particletypes of pulmonary delivery. The crystalline or glassy polymorphic formsof the 9-nitrocamptothecin is more stable over time than preparationswherein the 9-nitrocamptothecin molecules do not comprise a solid orquasi-solid, as when the 9-nitrocamptothecin molecules are solvated. Byway of example rather than limitation, any crystalline polymorph9-nitrocamptothecin can be used in a dry powder formulation forpulmonary delivery if it has been crystallized in microcrystalline form.Alternatively crystalline polymorphs of 9-nitrocamptothecin having maybe ground or pulverized to obtain a sufficiently small particle size,which may render them a corresponding polymorph having increasedamorphous content, or predominantly amorphous precipitate from rapidevaporation of solvent may be ground into a powdered glass form.

[0133] Dry powder formulations for pulmonary delivery include thecrystalline or amorphous polymorph and any carrier suitable forpulmonary drug administration, although pharmaceutical sugars aregenerally preferred as carriers, e.g., fructose, galactose, glucose,lactitol, lactose, maltitol, maltose, mannitol, melezitose, myoinositol,palatinite, raffinose, stachyose, sucrose, trehalose, xylitol, andhydrates and combinations thereof. Selected components are initiallycombined and then blended to form a homogeneous, uniform powder mixture.Techniques for preparation of such powders are well known in the art;briefly, the preparation typically includes the steps of reducing theparticle size of each component (as necessary), combining the individualcomponents and blending. Techniques of reducing the particle sizeemploy, by way of example, mills such as an air-jet mill or ball mill.Particle sizes having a diameter of between about 0.1 μm to about 65 μmare required for pulmonary administration. Blending methods includepassing the combined powders through a sifter and blending theindividual powders in a powder blender such as a “double cone” blenderor a “V-blender.” Regardless of the specific technique employed theresulting powder must be both homogeneous and uniform. Typically, theactive agents will make up from about 0.10% to about 99% (w/w) of thetotal formulation.

[0134] Pulmonary formulations of the present invention may also beadministered as aerosol compositions. Aerosol formulations are known tothose skilled in the art and described, for example, in Remington'sPharmaceutical Sciences, 19^(th) Ed. (Easton, Pa.: Mack PublishingCompany, 1995). Briefly, the aerosol formulation of the invention iseither a solution aerosol, in which the active agents are soluble in thecarrier (e.g., propellant), or a dispersion aerosol, in which the activeagents are suspended or dispersed throughout the carrier or carriers andoptional solvent. In aerosol formulations, the carrier is typically apropellant, usually a liquefied gas or mixture of liquified gases. Forexample, the carrier may be a fluorinated hydrocarbon. Preferredfluorinated hydrocarbons are selected from trichloromonofluoromethane,dichlorodifluoromethane, dichlorotetrafluoroethane,chloropentafluoroethane, 1-chloro-1,1-difluoroethane,1,1_difluoroethane, octafluorocyclobutane, 1,1,1,2-tetrafluoroethane(HFA-134a), 1,1,1,2,3,3,3-heptafluoropropane (HFA-227) and combinationsthereof. As is readily appreciated by one skilled in the art, theaerosol formulations of the invention may include one or moreexcipients. The aerosol formulations may, for example, contain: anantioxidant (e.g., ascorbic acid) for inhibiting oxidative degradationof the active agents; a dispersing agent (e.g., sorbitan trioleate,oleyl alcohol, oleic acid, lecithin, corn oil, and combinations thereof)for preventing agglomeration of particles; and/or a lubricant (e.g.,isopropyl myristate) for providing slippage between particles andlubricating the components, e.g., the valve and spring, of the inhaler.

[0135] As described with respect to the dry powder formulations, theparticle size released from aerosol formulations must be appropriate forpulmonary administration. Solution aerosols inherently produce smallparticles upon actuation of the inhaler because the active agent isexpelled along with the carrier, i.e., propellant, solution as itevaporates. Consequently, solution aerosol administration producessufficiently small particles, e.g., within a range of about 0.1 μm toabout 65 μm, of active agents. The crystalline and amorphous polymorphsof 9-nitrocamptothecin of the invention may only be delivered viaaerosol as a dispersion of solid in a liquid carrier.

[0136] Dispersion aerosols contain undissolved active agents in whichparticle size remains constant, i.e., the size of the particles in thedispersion aerosol remains unchanged during delivery of the activeagent. The active agents must therefore have an appropriate particlesize before formulation into a dispersion aerosol. Thus, techniques forreducing the particle size of active agents as described above for thedry powder formulations are equally applicable for preparing activeagents having an appropriate particle size in a dispersion aerosol.Further, the same ranges of particle sizes preferred for the dry powderformulations are applicable to dispersion aerosols.

[0137] Aerosol formulations of the invention may be prepared byutilizing a cold filling process. First, the components of the aerosolformulation and an aerosol container are cooled to about −40° C., sothat the carrier, i.e., propellant, is a liquid. All the componentsexcept for the carrier are then placed into the aerosol container. Next,the carrier is added and the components are mixed. A valve assembly isthen inserted into place. Finally, the valve assembly is crimped so thatthe container is airtight. The assembled container bearing the inhalantformulation may be allowed to return to ambient temperature afterassembly. As an alternative to the cold filling process, the aerosolformulation may be prepared by transfer of a carrier from a bulkcontainer after all the components except for the carrier are placedinto an aerosol container and a valve assembly is then inserted andcrimped into place. The liquid carrier is then metered under pressurethrough the valve assembly from a bulk container or tank. After thecarrier is metered in, the container is checked to ensure that thepressurized contents do not leak. For both of these methods of preparingaerosol formulations, the active agent will typically make up from about0.1 wt. % to about 40 wt. % of the total formulation. Preferably theactive agents make up about 1 wt. % to about 15 wt. % of the totalformulation.

[0138] The pulmonary formulations of the present invention may also be aliquid composition for inhalation, as is well known in the art. See,e.g., Remington: The Science and Practice of Pharmacy, supra. For the9-nitrocamptothecin polymorphs of the instant invention, the liquidcomposition must be a microsuspension. Such liquid formulations includeone or more carriers in addition to the active agents. As mentionedabove, care must be taken that a carrier does not solvate the polymorphis employed. An example of a carrier is a sodium chloride solutionhaving concentration making the formulation isotonic relative to normalbody fluid. In addition to the carrier, the liquid formulations maycontain water and/or excipients including an antimicrobial preservative(e.g., benzalkonium chloride, benzethonium chloride, chlorobutanol,phenylethyl alcohol, thimerosal and combinations thereof), a bufferingagent (e.g., citric acid, potassium metaphosphate, potassium phosphate,sodium acetate, sodium citrate, and combinations thereof), a surfactant(e.g., polysorbate 80, sodium lauryl sulfate, sorbitan monopalmitate andcombinations thereof), and/or a suspending agent (e.g., agar, bentonite,microcrystalline cellulose, sodium carboxymethylcellulose, hydroxypropylmethylcellulose, tragacanth, veegum and combinations thereof). Combiningthe components followed by conventional mixing effects a liquidformulation suitable for inhalation. Typically, the active agents willmake up from about 0.01% to about 40% of the total formulation.

[0139] Various known devices may be used to administer pulmonaryformulations, whether dry powder, aerosol or liquid. Dry powder inhalersare well known to those skilled in the art and are used to administerthe aforementioned dry powder formulations. Suitable dry powderinhalation devices for administering the present formulations include,for example, TURBOHALER® (Astra Pharmaceutical Products, Inc.,Westborough, Mass.), ROTAHALER® (Allen & Hanburys, Ltd., London,England). Aerosol formulations may be administered via pressurizedmetered-dose inhalers. Liquid formulations of the invention may beadministered via a pump spray bottle or nebulizer.

[0140] Other active agents may also be included in the formulations ofthe invention, including other anti-proliferative, anti-neoplastic oranti-inflammatory or bronchodilating agents that dilate the airway andeffect deeper delivery, especially for pathologies involvinginflammation of the bronchi or alveoli, or airway obstruction, forexample lung and broncoalveolar carcinomas. Agents that perform boththese functions, such as long acting β adrenergic agonists, includingsalmeterol xinafoate, and phosphodiesterase inhibitors, includingtheophylline and other hypoxanthines, have been shown to exert asynergistic anti-inflammatory effect in inflammatory pathohysiologicprocesses in the lung by Pang et al. (2000) Am. J. Respir. Cell Mol.Biol. 23(1):79-85.

[0141] Examples of suitable additional active agents to becoadministered with 9-nitrocamptothecin in the treatment ofproliferative respiratory disorders involving inflammation and/orobstruction include, without limitation, bronchodilators, including βadrenergic agonists, anticholinergics, phosphodiesterase inhibitorssuitable for inhalation, and corticosteroids. Combinations ofbronchodilators may also be used. Long acting β adrenergic agonists areparticularly preferred, as they will not only provide anti-inflammatoryeffects that often important in treating neoplastic pathologies of therespiratory system, but may also effect deeper delivery into the lung;this is especially important for lung and bronchoalveolar carcinomasinvolving alveolar inflammation. Likewise, any glucocorticoidtherapeutically suitable for administration by inhalant or apharmaceutically suitable salt ester or other derivative thereof may beincluded for co-administration by inhalant.

[0142] As alluded to above, bronchodilators are useful to ensuredelivery of active agent deep into the lungs. Typical bronchodilators ofthe anticholinergic type include, by way of example rather thanlimitation, atropinic compounds such as isatropium, which have beenshown to be strongly synergistic (Dusser (1998) Ann. Fr. Anesth. Reanim.17(Suppl. 2):40s-42s) with β agonists, specifically β₂ agonists, inbronchodilation for acute asthma and are expected to exert similareffects when used to open the airways to ensure deep delivery to thealveoli for delivery of anti-inflammatory agent. Typical bronchodilatorsof the β adrenergic agonist class include, but are not limited to,albuterol, bitolterol, clenbuterol, fenoterol, formoterol, levalbuterol(i.e., homochiral (R)-albuterol), metaproterenol, pirbuterol,procaterol, reproterol, rimiterol, salmeterol and terbutaline. Thebronchodilator may be present in the formulation as a salt, ester,amide, prodrug, or other derivative, or may be functionalized in variousways as will be appreciated by those skilled in the art.

[0143] Other anti-inflammatory drugs can be combined with9-nitrocamptothecin. Corticosteroids and non-steroidal anti-inflammatorydrugs (NSAIDS) are potential combinatorial therapy agents, and alreadyused in the treatment of inflammatory airway disease and neoplasms ingeneral. Cromolyn sulfate and the new class of leukotriene inhibitorsare also used in treating inflammatory disease, and may therefore beemployed inconjunction with the 9-nitrocamptothecin crystalline andamorphous polymorphs for inhalation therapy of both neoplasms associatedwith inflammation and primary inflammatory proliferative lungpathologies. Agents that are not primarily anti-inflammatory which havebeen evidenced to have anti-inflammatory activity include the longacting agonists and theophylline, as noted above, and macrolideantibiotics (Cazzola et al. (2000) Monaldi Arch. Chest Dis.55(3):231-6), which include erythromycin and its derivatives, e.g.,azithromycin and clarithromycin. Co-administration of antibiotics,including those with anti-inflammatory activity, or anti-viral agents,with the crystalline and amorphous polymorphs of the instant inventionis desirable for treatment of pulmonary neoplasias, which predispose thelungs to infection, and for treating, proliferative inflammatorydiseases of infectious etiology, such as pulmonary tuberculosis andviral pneumonitis.

[0144] C. Transdermal Administration

[0145] Particulate suspensions, microsuspensions and nano suspensions aswell as emulsifications of various particulate sizes, including theparticulate sizes appropriate for pulmonary administration may beconverted to transdermal delivery of 9-nitrocamptothecin. Alternativelylarger size crystalline and/or amorphous polymorphs of the invention maybe formulated as an emulsified, including microemulsified, dispersion,with addition of an appropriate emulsifying agent. However theparticulate sizes obtained for pulmonary administration may be directlycombined with an appropriate agent that preserves the particles whilepermitting the diffusion of 9-nitrocamptothecin molecules therethroughand transdermally upon application through the skin

[0146] 3. Indications

[0147] The 9-nitrocamptothecin polymorphs may be used to treat anydisease state in which 9-nitrocamptothecin is therapeutically effective.In order to take advantage of the novel polymorphs of the presentinvention, the pharmaceutical formulations in which the polymorphs areincorporated and administered should retain their polymorphic form.

[0148] According to one embodiment, a method is provided for treating adisease state comprising administering to a patient a formulationcomprising one or more 9-nitrocamptothecin polymorphs.

[0149] In one variation, a formulation comprising the9-nitrocamptothecin polymorph is administered to a patient having adisease state associated with an undesirable or uncontrolled cellproliferation. Such indications include, for example, restenosis (e.g.coronary, carotid, and cerebral lesions), benign tumors, various typesof cancers such as primary tumors and tumor metastases, abnormalstimulation of endothelial cells (atherosclerosis), insults to bodytissue due to surgery or other events leading to formation of scartissue, abnormal wound healing, abnormal angiogenesis, diseases thatproduce fibrosis of tissue, repetitive motion disorders, disorders oftissues that are not highly vascularized, proliferative responsesassociated with organ transplants and various inflammatory proliferativediseases.

[0150] Generally, cells in a benign tumor retain their differentiatedfeatures and do not divide in a completely uncontrolled manner. A benigntumor is usually localized and nonmetastatic. Specific types of benigntumors that can be treated using the present invention include, withoutlimitation, hemangiomas such as cavernous hemangioma, hepatocellularadenoma, cavernous hemangioma, focal nodular hyperplasia, acousticneuromas, neurofibroma, bile duct adenoma, bile duct cystanoma, fibroma,lipomas, benign bone tumors, leiomyomas, mesotheliomas, teratomas,myxomas, nodular regenerative hyperplasia, trachomas and granulomatousinflammatory diseases both infectious, such as pyogenic granulomas, andnon-infectious or idiopathic, such as sarcoidosis and berylliosis.

[0151] In a neoplasia such as a malignant tumor, cells becomeundifferentiated, do not respond to physiologic cell proliferationcontrol signals, and multiply in an uncontrolled manner. The malignanttumor is invasive and capable of spreading to distant sites(metastasizing). Malignant tumors and other neoplasias may usually bedivided into primary and secondary neoplasias. A primary neoplasiaarises directly from the tissue of origin and may spread to contiguoustissues and organs by local invasion. A secondary neoplasia, ormetastasis, is exemplified by a tumor that originated elsewhere in thebody but has now spread to a distant organ. The common routes for spreadof neoplasia are direct growth into adjacent structures, and metastaticspread through the vascular or lymphatic systems, and tracking alongtissue planes and body spaces including peritoneal fluid, cerebrospinalfluid, etc.

[0152] Specific types of cancers or neoplasias, both primary andsecondary, that can be treated using this invention include bothcarcinomas and sarcomas. Examples of specific carcinomas and sarcomasinclude leukemia, breast cancer, skin cancer, bone cancer, prostatecancer, liver cancer, lung cancer, neurological tumors of the brain,cancer of the larynx, gallbladder, pancreas, rectum, parathyroid,thyroid, adrenal, neural tissue, head and neck, colon, stomach, bronchi,kidneys, basal cell carcinoma, squamous cell carcinoma of bothulcerating and papillary type, metastatic skin carcinoma, osteosarcoma,Ewing's sarcoma, reticulum cell sarcoma, myeloma, giant cell tumor,small-cell lung tumor, gallstones, islet cell tumor, primary braintumor, acute and chronic lymphocytic and granulocytic tumors, hairy-celltumor, adenoma, hyperplasia, medullary carcinoma, pheochromocytoma,mucosal neuromas, intestinal ganglioneuromas, hyperplastic corneal nervetumor, marfanoid habitus tumor, Wilm's tumor, seminoma, ovarian tumor,leiomyomas, cervical dysplasia and other in situ carcinomas,neuroblastoma, retinoblastoma, soft tissue sarcoma, malignant carcinoid,topical skin lesion, mycosis fungoides, rhabdomyosarcoma, Kaposi'ssarcoma, osteogenic and other sarcomas, malignant hypercalcemia, renalcell tumor, polycythemia vera, adenocarcinomas, glioblastoma multiforma,leukemias, lymphomas, melanoma, epidermoid carcinomas.

[0153] Treatment of abnormal cell proliferation due to insults to bodytissue during surgery may be possible for a variety of surgicalprocedures, including joint surgery, bowel surgery, and keloid scarring.Diseases that produce fibrotic tissue include emphysema. Repetitivemotion disorders that may be treated using the present invention includecarpal tunnel syndrome.

[0154] The proliferative responses associated with organ transplantationthat may be treated using this invention include those proliferativeresponses contributing to potential organ rejections or associatedcomplications. Specifically, these proliferative responses may occurduring transplantation of the heart, lung, liver, kidney, and anyforeign or non-self cells, tissues, organs or organ systems.

[0155] Abnormal angiogenesis that may be may be treated using thisinvention include those abnormal angiogenesis accompanying rheumatoidarthritis, ischemic-reperfusion related brain edema and injury, adrenalcortical ischemia, ovarian hyperplasia and hypervascularity, polycysticovary syndrome, endometriosis, psoriasis, diabetic retinopaphy, andother ocular angiogenic diseases such as retinopathy of prematurity(retrolental fibroplastic disease), macular degeneration, corneal graftrejection, neuroscular glaucoma and Oster Webber syndrome.

[0156] Diseases associated with abnormal angiogenesis require or inducevascular growth. For example, corneal angiogenesis involves threephases: a pre-vascular latent period, active neovascularization, andvascular maturation and regression. The identity and mechanism ofvarious angiogenic factors, including elements of the inflammatoryresponse, such as leukocytes, platelets, cytokines, and eicosanoids, orunidentified plasma constituents have yet to be revealed.

[0157] In another embodiment of the present invention, a method isprovided for treating diseases associated with undesired anduncontrolled angiogenesis. The method comprises administering to apatient suffering from uncontrolled angiogenesis a therapeuticallyeffective amount of 9-nitrocamptothecin, such that formation of bloodvessels is inhibited. The particular dosage of 9-nitrocamptothecinrequired to inhibit angiogenesis and/or angiogenic diseases may dependon the severity of the condition, the route of administration, andrelated factors that can be decided by the attending physician.Generally, accepted and effective daily doses are the amount sufficientto effectively inhibit angiogenesis and/or angiogenic diseases.

[0158] According to this embodiment, the composition of the presentinvention may be used to treat a variety of diseases associated withuncontrolled angiogenesis such as retinal/choroidal neovascularizationand corneal neovascularization. Examples of retinal/choroidalneovascularization include, without limitation, Best's disease, myopia,optic pits, Stargart's disease, Paget's disease, vein occlusion, arteryocclusion, sickle cell anemia, sarcoid, syphilis, pseudoxanthomaelasticum carotid abostructive diseases, chronic uveitis/vitritis,mycobacterial infections, Lyme disease, systemic lupus erythematosis,retinopathy of prematurity, Eale's disease, diabetic retinopathy,macular degeneration, Behcet's disease, infections causing a retinitisor choroiditis, ocular histoplasmosis, pars planitis, chronic retinaldetachment, hyperviscosity syndromes, toxoplasmosis, trauma andpost-laser complications, diseases associated with rubesis(neovascularization of the angle) and diseases caused by the abnormalproliferation of fibrovascular or fibrous tissue including all forms ofproliferative vitreoretinopathy. Examples of corneal neuvascularizationinclude, but are not limited to, epidemic keratoconjunctivitis, VitaminA deficiency, contact lens overwear, atopic keratitis, superior limbickeratitis, pterygium keratitis sicca, Sjogren's syndrome, acne rosacea,phylectenulosis, diabetic retinopathy, retinopathy of prematurity,corneal graft rejection, Mooren ulcer, Terrien's marginal degeneration,marginal keratolysis, polyarteritis, Wegener granulomatosis,sarcoidosis, scleritis, pemphigoid, radial keratotomy, neovascularglaucoma and retrolental fibroplasia, syphilis, Mycobacteria infections,lipid degeneration, chemical burns, bacterial ulcers, fungal ulcers,Herpes simplex infections, Herpes zoster infections, protozoaninfections and Kaposi sarcoma.

[0159] In yet another embodiment of the present invention, a method isprovided for treating chronic inflammatory diseases associated withuncontrolled angiogenesis. The method comprises administering to apatient suffering from a chronic inflammatory disease associated withuncontrolled angiogenesis a therapeutically effective amount of thecomposition of the present invention, such that formation of bloodvessels is inhibited. The chronic inflammation depends on continuousformation of capillary sprouts to maintain an influx of inflammatorycells. The influx and presence of the inflammatory cells producegranulomas and thus maintains the chronic inflammatory state. Inhibitionof angiogenesis using the composition of the present invention alone orin conjunction with other anti-inflammatory agents may prevent theformation of the granulomas, thereby alleviating the disease. Examplesof chronic inflammatory disease include, but are not limited to,inflammatory bowel diseases such as Crohn's disease and ulcerativecolitis, psoriasis, sarcoidosis, and rheumatoid arthritis.

[0160] Inflammatory bowel diseases such as Crohn's disease andulcerative colitis are characterized by chronic inflammation andangiogenesis at various sites in the gastrointestinal tract. Forexample, Crohn's disease occurs as a chronic transmural inflammatorydisease that most commonly affects the distal ileum and ascending colonbut may also occur in any part of the gastrointestinal tract from themouth to the anus and perianal area. Patients with Crohn's diseasegenerally have chronic diarrhea associated with abdominal pain, fever,anorexia, weight loss and abdominal swelling. Ulcerative colitis is alsoa chronic, nonspecific, inflammatory and ulcerative disease arising inthe colonic mucosa and is characterized by the presence of bloodydiarrhea.

[0161] These inflammatory bowel diseases are generally caused by chronicgranulomatous inflammatory pathophysiologic processes. Inflammatorybowel disease may affect the entire gastrointestinal tract, typicallyinvolving new capillary sprouts surrounded by a cylinder of inflammatorycells. Inhibition of angiogenesis by the composition of the presentinvention should inhibit the formation of the sprouts and prevent theformation of granulomas. The inflammatory bowel diseases also exhibitextra intestinal manifestations, such as skin lesions. Such lesions arecharacterized by inflammation and angiogenesis and can occur at manysites other the gastrointestinal tract. Inhibition of angiogenesis bythe composition of the present invention should reduce the influx ofinflammatory cells and prevent, halt or slow pathogenesis of the lesion.

[0162] Sarcoidois, another chronic inflammatory disease, ischaracterized as an idiopathic multisystem granulomatous disorder.Berylliosis resembles sarcoidosis histopathologically, but is known tobe caused by the element Beryllium. The granulomas of sarcoidosis andberylliosis histopathologically resemble the non-caseating granulomas ofMycobacterium tuberculosis and other diseases caused by Mycobacteria,but caseating granulomas found in M. Tuberculosis infection are absentin both berylliosis and sarcoidosis. The granulomas of this disease canform anywhere in the body and, thus, the symptoms depend on the site ofthe granulomas and whether the disease is active. The formation ofsarcoid granulomas is facilitated by the angiogenic capillary sprouts,which provide a constant supply of inflammatory cells. By using thecomposition of the present invention to inhibit angiogenesis, suchgranuloma formation can be inhibited.

[0163] Psoriasis, also a chronic and recurrent inflammatory disease, ischaracterized by papules and plaques of various sizes. Treatment usingthe composition of the present invention alone or in conjunction withother anti-inflammatory agents should prevent the formation of new bloodvessels necessary to maintain the characteristic lesions and provide thepatient relief from the symptoms.

[0164] Rheumatoid arthritis (RA) is also a chronic inflammatory diseasecharacterized by non-specific inflammation of the peripheral joints. Itis believed that the blood vessels in the synovial lining of the jointsundergo angiogenesis. In addition to forming new vascular networks, theendothelial cells release factors and reactive oxygen species that leadto pannus growth and cartilage destruction. The factors involved inangiogenesis may actively contribute to, and help maintain, thechronically inflamed state of rheumatoid arthritis. Treatment using thecomposition of the present invention alone or in conjunction with otheranti-RA agents should prevent the formation of new blood vesselsnecessary to maintain the chronic inflammation and provide the RApatient relief from the symptoms.

[0165] The composition of the present invention may also be used inconjunction with other anti-angiogenesis agents to inhibit undesirableand uncontrolled angiogenesis. Examples of anti-angiogenesis agentsinclude, but are not limited to, retinoic acid and derivatives thereof,2-methoxyestradiol, ANGIOSTATIN™ protein, ENDOSTATIN™ protein, suramin,squalamine, tissue inhibitor of metalloproteinase-I, tissue inhibitor ofmetalloproteinase-2, plasminogen activator inhibitor-1, plasminogenactivator inhibitor-2, cartilage-derived inhibitor, paclitaxel, plateletfactor 4, protamine sulphate (clupeine), sulphated chitin derivatives(prepared from queen crab shells), sulphated polysaccharidepeptidoglycan complex (sp-pg), staurosporine, modulators of matrixmetabolism, including for example, proline analogs((1-azetidine-2-carboxylic acid (LACA), cishydroxyproline,d-1,3,4-dehydroproline, thiaproline], α, α-dipyridyl,β-aminopropionitrile fumarate, 4-propyl-5-(4-pyridinyl)-2(3h)-oxazolone;methotrexate, mitoxantrone, heparin, interferons, 2 macroglobulin-serum,chimp-3, chymostatin, β-cyclodextrin tetradecasulfate, eponemycin,fumagillin, gold sodium thiomalate, d-penicillamine (CDPT),β-1-anticollagenase-serum, α-2-antiplasmin, bisantrene, lobenzaritdisodium, n-(2-carboxyphenyl-4-chloroanthronilic acid disodium or “CCA”,thalidomide; angiostatic steroid, carboxyaminoimidazole;metalloproteinase (metalloprotease) inhibitors such as BB94. Otheranti-angiogenesis agents include antibodies, preferably monoclonalantibodies against these angiogenic growth factors: bFGF, aFGF, FGF-5,VEGF isoforms, VEGF-C, HGF/SF and Ang-1/Ang-2. Ferrara N. and Alitalo,K. “Clinical application of angiogenic growth factors and theirinhibitors” (1999) Nature Medicine 5:1359-64.

[0166] In all embodiments, the term “effective amount” is understood asa medical art term, that is, the dose schedule and route ofadministration of the drug that gives the best therapeutic value andconvenience to the patient.

EXAMPLES

[0167] The following examples set forth methods for analyzing thecompositions of the present invention, studies regarding thecharacterization of their physical and chemical properties and methodsof preparing the formulations according to the present invention.

[0168] 1. Preparation of Samples

[0169] Three samples of 9-nitrocamptothecin were received from SuperGenPharmaceutical Research Institute. A representative XRPD patternexhibited by these samples is provided in FIG. 1. The polymorphic formgiving this pattern is designated Form A.

[0170] 2. Characterizing Stability in a Solvent by Interconversion ofForms

[0171] The initial material characterized was designated Form A and wasisolated by direct crystallization of 9-nitrocamptothecin from anacetone/ethanol solvent mixture. Form B was obtained from either acetoneor dichloromethane and appears to be a hemihydrate. Form C appears to bea mono tetrahydrofuran (THF) solvate. Form D, which was obtained bycrystallization from an acetonitrile based solvent, appears to be anacetonitrile mono-solvate. Form E was which was obtained bycrystallization from a chloroform based solvent, appears to be a ¼equivalent chloroformic solvate. Form F has been obtained byinterconversion from certain polymorphs, notably Forms B, C and D,typically in a dimethylformamide and water (75%/25%, v/v), see supra.Form F has also been obtained by crystallization from acetone/H₂O(80%/20%, v/v). Form G was obtained by direct crystallization of9-nitrocamptothecin from dimethylformamide and appears to be asolvate/hydrate.

[0172] A. Characterizing Solubility

[0173] A weighed sample of 9-nitrocamptothecin (typically 10 to 20 mg)was treated with aliquots of the test solvent. Solvents were eitherreagent or HPLC grade. The aliquots were typically either 150 μL or 1mL. Between additions the mixture was typically shaken or sonicated.Whether the solids dissolved was judged by visual inspection, anapproximate solubility was obtained. These approximate solubilities areprovided below in Table 15.

[0174] The solution was filtered followed by one of several differentfinal processing steps, which are reported below as conditions in Table16. TABLE 15 Approximate Solubilities of 9-nitrocamptothecinSolvent^(a,b) Solubility (mg/mL)^(c) Notebook No. Acetone <1 294-63-01acetonitrile (ACN) <2 294-63-07 Dichloromethane 2 294-61-02dimethylformamide (DMF) 8 294-65-02 Ethanol <3 294-63-02 Ethylacetate <3294-63-04 Hexanes <2 294-65-03 Methanol <2 294-61-03 methyl ethyl ketone(MEK) <3 294-64-02 tetrahydrofuran (THF) <2 294-63-06 Toluene <2294-64-01 Water <4 294-63-03

[0175] TABLE 16 Polymorph Screen of 9-nitrocamptothecin Sample SourceConditions^(a) Habit^(b) Lot no. 99-064 as received — (SSCI no. 8499)Lot no. 99-064 as received — (SSCI no. 8500) Lot no. 99-064 as received— (SSCI no. 8501) Lot no. 99-064 10′ grind Nd Lot no. 99-064 melt blacksolids Acetone FE Needles SE Needles SC (45° C.) Needles SC (45° C.)Needles SC (45° C.) Needles SC (45° C.) Needles SC (45° C.) Needles SC(45° C.) Needles SC (45° C.) Nd SC (45° C.) Nd acetone/ethanol (1:1) SL,RT, 3 hrs Nd SL, 40° C., 3 hrs Nd SL, reflux, 3 hrs Nd Acetonitrile FEUnknown (ACN) SH Rods SH needles/rods SH needles/rods SH needles/rods SHNd SH Nd SC (60° C.) plates, rods Chloroform FE Needles FE Needles FENeedles FE Needles SE Needles SE Nd SE Nd SC (45° C.) NeedlesDichloromethane FE Needles (DCM) FE Nd SE Needles SC (45° C.) Needles SC(45° C.) Needles SC (45° C.) Needles RV (50° C.) Unknown RV (50° C.) NdDimethylformamide FE Unknown SE Needles SC (60° C.) Unknown Ethanol FEUnknown SH needles, rods SC (60° C.) Needles Ethylacetate FE Needles(EtOAc) SH Rods SC (60° C.) Needles Heptane FE no solids SC (60° C.) NdHexanes FE no solids SH needles, rods SC (60° C.) Unknown Methanol FEUnknown SH needles, rods SC (60° C.) Needles RV (50° C.) Unknown RV (50°C.) Unknown RV (50° C.) Unknown Methylethylketone FE Needles (MEK) SHRods SC (60° C.) Needles Tetrahydrofuran FE Unknown (THF) FE Unknown FEUnknown FE Unknown FE Nd SE Nd SC (60° C.) Unknown Toluene FE Needles SHneedles, rods SC (60° C.) Needles Water FE Unknown (H₂O) SH needles,rods SC (60° C.) unknown paste

[0176] Fast evaporation (FE) indicates the filtered solution was left inan open vial under ambient conditions. Slow evaporation (SE) indicatesthe resulting solution was left under ambient conditions in a vialcovered with aluminum foil containing pinholes. In some cases, thesolvent was removed using a rotary evaporator (RV) with the sample bathtemperature at 50° C.

[0177] The same procedure was repeated at elevated temperature (45 or60° C.) by keeping the mixture on a hot plate at the desiredtemperature. The resulting solution was rapidly filtered into a vialkept on the same hot plate. The heat source was turned off and the hotplate and vial were allowed to cool to ambient temperature. The vial wasthen allowed to stand at ambient temperature overnight. The presence orabsence of solids was noted. If sufficient solids were present, thesolution was filtered and the solids collected. If insufficient solidswere present, the vial was placed in a refrigerator overnight. Again thepresence or absence of solids was noted and, if there were none, thevial was placed in a freezer overnight. Solids were removed byfiltration and allowed to dry in the air.

[0178] Solubilities were estimated from these experiments based on thetotal solvent used to give a solution. Duplicate runs were averaged. Theactual solubilities may be greater than those calculated due to the sizeof the solvent aliquots used, or due to a slow rate of dissolution. Ifdissolution did not occur during the experiment the solubility isexpressed as “less than.”

[0179] 9-nitrocamptothecin slurries were agitated for 3 to 4 days in ashaker block at ambient temperature (slurry). Solids were removed byfiltration and allowed to dry in the air. The remaining clear, yellowsolutions were left for fast evaporation.

[0180] Crystallizations by addition of anti-solvent were performed byfiltering a saturated solution of 9-nitrocamptothecin in DMF dropwiseinto 5 ml of an antisolvent. The antisolvents employed for thecrystallization experiments included acetone, ethyl acetate, methylethyl ketone, toluene, and water. If solid precipitate was not presentafter the dopwise addition through a filter, the solution was capped andrefrigerated. The sample was monitored periodically after beginningrefrigeration, and if precipitate appeared, it was removed by filtrationand allowed to air dry.

[0181] Hygroscopicity was investigated by placing a sample in a sealedchamber at room temperature and 95% relative humidity for 20 days.Weight gain/loss or TGA were not measured in the course of this study ofhygroscopicity. An XRPD pattern was obtained on the solid remainingafter 20 days and compared to the starting material.

[0182] Dehydration/desolvation studies were conducted by placing asample under continuous vacuum at room temperature for 14 days. An XRPDpattern was obtained on the remaining solid and compared to the startingmaterial.

[0183] A solidified melt of 9-nitrocamptothecin was produced by slowlyheating the sample on a hot bench until a visual melt was observed andthen quickly cooling the sample to ambient temperature. As the materialbegan to melt, it turned dark and bubbled. The resulting dark materialwas not analyzed further due to decomposition.

[0184] 9-nitrocamptothecin as received, e.g. Form A, was ground in anamalgamator for 10 minutes. The sample was then analyzed using XRPD. Theobserved XRPD pattern of the ground Form A was that of Form A withnoticeable line broadening indicative of an increased proportion ofglassy material relative to the unground Form A.

[0185] B. Characterizing Solubility and Interconversion of Forms

[0186] Interconversion experiments were carried out using Forms A, B, C,D, and E in two solvent systems, toluene and dimethylformamide/water(75%/25%, v/v). The experimental data indicate that in toluene, Form Ais the most stable polymorph and Form B is more stable than Form C orForm E. In the DMF/H2O solvent mixture, Form F appears to be the moststable hydrated form, but form A was also isolated from this solventmixture.

[0187] These interconversion experiments were carried out by makingslurries containing two or more forms in saturated toluene or DMF/water(75/25) solutions. The slurries were agitated for 7 days at ambienttemperature either in a shaker block or on a rotating wheel. Theinsoluble solids present after the agitation were recovered byfiltration and analyzed using XRPD.

[0188] 3. Characterization

[0189] A. X-Ray Powder Diffraction

[0190] X-ray powder diffraction analyses were carried out on a ShimadzuXRD-6000 X-ray powder diffractometer using Cu Kα radiation having awavelength of 1.5406 Å. The instrument is equipped with a fine-focusX-ray tube. The tube power was set by setting potential difference at 40kV, and current 40 mA. The divergence and scattering slits were set at1° and the receiving slit was set at 0.15 mm. Diffracted radiation wasdetected by a NaI scintillation detector. A theta-two theta continuousscan at 3°/min (0.4 sec/0.02° step) from 2.5 to 40 °2θ was performed. Asilicon standard was analyzed each day to check the instrumentalignment. Each sample was analyzed in a quartz sample holder. Avariable temperature (VT-XRPD) experiment was performed on one form. Thesample was prepared for analysis by pressing it into a variabletemperature holder. A powder pattern was collected initially at 35° C.and again, after heating, at 200° C.

[0191] B. Thermo and Thermogravimetric Analysis

[0192] Thermogravimetric analysis (TGA) was carried out on a TAInstruments TGA 2050. The calibration standards were nickel and alumel.Approximately 2-7 mg of sample was placed on a clean, platinum pan,accurately weighed, and inserted into the TGA furnace. The samples wereheated in nitrogen at a rate of 10° C./min, from 35° C. to a finaltemperature of 375° C.

[0193] DSC data were obtained on a TA Instruments DSC 2920. Thecalibration standard was indium. Approximately 1.5-2.5 mg of sample wasplaced into a DSC pan, and the weight accurately recorded. The pans werehermetically sealed with a pinhole to allow for pressure release. Notethat the observed volatilization temperatures may be higher than thoseobtained in open pans due to pressure effects. The samples were heatedunder nitrogen at a rate of 10° C. min, from 25° C. to a finaltemperature of 350° C.

[0194] Hot-stage microscopy was carried out using a Wagner & Munzapparatus consisting of a Kofler stage mounted on a Leica Microscope.The stage temperature was calibrated using vanillin and caffeine USPstandards each day prior to running samples. For each sample, a smallquantity was placed on a microscope slide and covered. Samples wereheated at approximately 4° C./min. and images were captured periodicallyusing a 10X objective lens and a CCD camera. A cross-polarizing filterwas used to observe birefringence.

[0195] C. Infrared Spectroscopy

[0196] Mid-IR spectra were acquired on a Nicolet model 860 Fouriertransform IR spectrophotometer equipped with a globar source, Ge/KBrbeamsplitter, and deuterated triglycine sulfate (DTGS) detector. ASpectra-Tech, Inc. diffuse reflectance accessory was utilized forsampling. Each spectrum represents 128 co-added scans at a spectralresolution of 2 cm⁻¹, except for sample 270-86-01, for which thespectrum was obtained with 256 scans at a resolution of 4 cm⁻¹. Abackground data set was acquired with an alignment mirror in place. Asingle beam sample data set was then acquired. Subsequently, a Log 1/R(R=reflectance) spectrum was acquired by taking the ratio of the twodata sets against each other. The spectrophotometer wavelength wascalibrated with polystyrene at the time of use.

[0197] D. Raman Spectroscopy

[0198] Raman spectra were acquired on a Raman accessory interfaced to aNicolet Magna 860 Fourier transform infrared spectrometer utilizing anexcitation wavelength of 1064 nm and approximately 0.5 W of Nd:YAG laserpower. A routine spectrum represents 128 co-added scans at a spectralresolution of 4 cm⁻¹. Each sample was prepared for analysis by placingit in a 5-mm diameter glass tube and positioning this tube in thespectrometer. The spectrometer was calibrated (wavelength) with sulfurand cyclohexane at the time of use.

[0199] E. NMR Spectroscopy

[0200] Solution state ¹H NMR data were obtained by Special Data Servicesof Champaign, Ill. using a 400 MHz Varian spectrometer. Approximately 15to 30 mg of each form was dissolved in d₆-DMSO, dried over molecularsieves, and placed in a 5 mm NMR tube, which had been dried at 250° C.for 6 hours. Data were collected at a ¹H resonant frequency of 399.798MHz, with a 7 kHz sweep width/filter, 32K data points, and 40acquisitions. Additional parameters included a 7 μs ¹H pulse width and a5 second pulse delay. The FID data was processed by zerofilling to 64Kdata points and multiplying by 0.2 Hz exponential line broadening priorto Fourier transformation. Predicted chemical shift values were based onthe structure using ChemDraw Pro.

[0201] F. Moisture Balance

[0202] Moisture sorption/desorption data were collected on a VTI SGA-100moisture balance system. For sorption isotherms, a sorption range of 5to 95% relative humidity (RH) and a desorption range of 95 to 5% RH in10% RH increments was used for analysis. The samples were not driedprior to analysis. Equilibrium criteria used for analysis were less than0.0100 weight % change in 5 minutes with a maximum equilibration time of3 hours if the weight criterion was not met. Data were not corrected forthe initial moisture content of the samples.

[0203] G. Karl Fischer Water Analysis

[0204] Karl Fischer (titrimetric) water analysis was performed byGalbraith Laboratories, Inc. of Knoxville, Tenn. according to U.S.Pharmacopoeia, vol. 24, method 921, U.S.P. Pharmacopeial Convention,Inc, Rockville, Md.). The polymorph was tested for water content by KarlFischer titration using a coulometer according to the publishedprocedure and the manufacturer's coulometer instructions.

[0205] It will be apparent to those skilled in the art that variousmodifications and variations can be made to the compounds, compositions,and methods of the present invention without departing from the spiritor scope of the invention. Thus, it is intended that the presentinvention cover the modifications and variations of this inventionprovided they come within the scope of the appended claims and theirequivalents.

We claim:
 1. A polymorphic form of 9-nitrocamptothecin, the polymorphbeing characterizable as having, by differential scanning calorimetry,no observable endotherm and an exotherm at between 273.6 and 275.6° C.,and a solution NMR spectrum with multiplets at 1.7 and 3.7 ppm shifts.2. A polymorphic form of 9-nitrocamptothecin according to claim 1, thepolymorph being further characterizable as having an exotherm bydifferential scanning calorimetry at between 274.1 and 275.1° C.
 3. Apolymorphic form of 9-nitrocamptothecin according to claim 1, thepolymorph being further characterizable as having an exotherm bydifferential scanning calorimetry at between 274.4 and 274.8° C.
 4. Apolymorphic form of 9-nitrocamptothecin according to claim 1, thepolymorph being further characterizable as having an exotherm bydifferential scanning calorimetry at between 274.5 and 274.7° C.
 5. Apolymorphic form of 9-nitrocamptothecin according to claim 1, whereinthe polymorph is obtained by grinding.
 6. A polymorphic form of9-nitrocamptothecin, the polymorph being characterizable as having anX-ray powder diffraction pattern with diffraction lines at °2θ values6.7, 12.5, 14.0 and 23.9 for Cu Kα radiation of wavelength 1.5406Angstrom.
 7. A polymorphic form of 9-nitrocamptothecin, the polymorphbeing characterizable as having an X-ray powder diffraction pattern withdiffraction lines at °2θ values 6.7, 12.5, 14.0 and 23.9 for Cu Kαradiation of wavelength 1.5406 Angstrom.
 8. A polymorphic form of9-nitrocamptothecin, the polymorph being characterizable as having, forCu Kα radiation of wavelength 1.5406 Angstrom, an X-ray powderdiffraction pattern with diffraction lines at °2θ values 6.7, 12.5, 14.0and 23.9.
 9. 9-nitrocamptothecin in a form crystallized fromtetrahydrofuran.
 10. A polymorphic form of 9-nitrocamptothecin accordingto claim 10, the polymorph being characterizable as having, bydifferential scanning calorimetry, no observable endotherm and anexotherm at between 273.6 and 275.6° C., and a solution NMR spectrumwith multiplets at 1.7 and 3.7 ppm shifts.
 11. A polymorphic form of9-nitrocamptothecin according to claim 10, the polymorph beingcharacterizable as having an X-ray powder diffraction pattern withdiffraction lines at °2θ values 6.7, 12.5, 14.0 and 23.9 for Cu Kαradiation of wavelength 1.5406 Angstrom.
 12. A polymorphic form of9-nitrocamptothecin according to claim 10, the polymorph beingcharacterizable as having an X-ray powder diffraction pattern withdiffraction lines at °2θ values 6.7, 12.5, 14.0 and 23.9 for Cu Kαradiation of wavelength 1.5406 Angstrom.
 13. A polymorphic form of9-nitrocamptothecin according to claim 10, the polymorph beingcharacterizable as having, for Cu Kα radiation of wavelength 1.5406Angstrom, an X-ray powder diffraction pattern with diffraction lines at°2θ values 6.7, 12.5, 14.0 and 23.9.
 14. A pharmaceutical compositioncomprising: a pharmaceutical carrier; and a polymorphic form of9-nitrocamptothecin, the polymorph being characterizable as having, bydifferential scanning calorimetry, no observable endotherm and anexotherm at between 273.6 and 275.6° C., and a solution NMR spectrumwith multiplets at 1.7 and 3.7 ppm shifts.
 15. A pharmaceuticalcomposition according to claim 14, the polymorph being furthercharacterizable as having an exotherm by differential scanningcalorimetry at between 274.1 and275.1° C.
 16. A pharmaceuticalcomposition according to claim 14, the polymorph being furthercharacterizable as having an exotherm by differential scanningcalorimetry at between 274.4 and 274.8° C.
 17. A pharmaceuticalcomposition according to claim 14, the polymorph being furthercharacterizable as having an exotherm by differential scanningcalorimetry at between 274.5 and 274.7° C.
 18. A pharmaceuticalcomposition comprising: a pharmaceutical carrier; and a polymorphic formof 9-nitrocamptothecin, the polymorph being characterizable as having anX-ray powder diffraction pattern with diffraction lines at °2θ values6.7, 12.5, 14.0 and 23.9 for Cu Kα radiation of wavelength 1.5406Angstrom.
 19. A pharmaceutical composition comprising: a pharmaceuticalcarrier; and a polymorphic form of 9-nitrocamptothecin, the polymorphbeing characterizable as having an X-ray powder diffraction pattern withdiffraction lines at °2θ values 6.7, 12.5, 14.0 and 23.9 for Cu Kαradiation of wavelength 1.5406 Angstrom.
 20. A pharmaceuticalcomposition comprising: a pharmaceutical carrier; and a polymorphic formof 9-nitrocamptothecin, the polymorph being characterizable as having,for Cu Kα radiation of wavelength 1.5406 Angstrom, an X-ray powderdiffraction pattern with diffraction lines at °2θ values 6.7, 12.5, 14.0and 23.9.
 21. A pharmaceutical composition comprising: a pharmaceuticalcarrier; and a polymorphic 9-nitrocamptothecin in a form crystallizedfrom tetrahydrofuran.
 22. A pharmaceutical composition according toclaim 21, the polymorph being characterizable as having, by differentialscanning calorimetry, no observable endotherm and an exotherm at between273.6 and 275.6° C., and a solution NMR spectrum with multiplets at 1.7and 3.7 ppm shifts.
 23. A pharmaceutical composition according to claim21, the polymorph being characterizable as having an X-ray powderdiffraction pattern with diffraction lines at °2θ values 6.7, 12.5, 14.0and 23.9 for Cu Kα radiation of wavelength 1.5406 Angstrom.
 24. Apharmaceutical composition according to claim 21, the polymorph beingcharacterizable as having an X-ray powder diffraction pattern withdiffraction lines at °2θ values 6.7, 12.5, 14.0 and 23.9 for Cu Kαradiation of wavelength 1.5406 Angstrom.
 25. A pharmaceuticalcomposition according to claim 21, the polymorph being characterizableas having, for Cu Kα radiation of wavelength 1.5406 Angstrom, an X-raypowder diffraction pattern with diffraction lines at °2θ values 6.7,12.5, 14.0 and 23.9.
 26. A method of preparing a polymorphic form of9-nitrocamptothecin, the method comprising: crystallizing9-nitrocamptothecin from tetrahydrofuran.
 27. A method according toclaim 26, the polymorph being characterizable as having, by differentialscanning calorimetry, no observable endotherm and an exotherm at between273.6 and 275.6° C., and a solution NMR spectrum with multiplets at 1.7and 3.7 ppm shifts.
 28. A method according to claim 26, the polymorphbeing characterizable as having an X-ray powder diffraction pattern withdiffraction lines at °2θ values 6.7, 12.5, 14.0 and 23.9 for Cu Kαradiation of wavelength 1.5406 Angstrom.
 29. A method according to claim26, the polymorph being characterizable as having an X-ray powderdiffraction pattern with diffraction lines at °2θ values 6.7, 12.5, 14.0and 23.9 for Cu Kα radiation of wavelength 1.5406 Angstrom.
 30. A methodaccording to claim 26, the polymorph being characterizable as having,for Cu Kα radiation of wavelength 1.5406 Angstrom, an X-ray powderdiffraction pattern with diffraction lines at °2θ values 6.7, 12.5, 14.0and 23.9.